Therapeutic uses for nitric oxide inhibitors

ABSTRACT

The present invention is based on the discovery that nitric oxide (NO) is an important growth regulator in an intact developing organism. In particular, the present invention relates to a method of increasing in a mammal a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue, wherein the hematopoietic tissue is contacted with at least one inhibitor of NO, such as one or more inhibitors of nitric oxide synthase (NOS), thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation. The present invention also relates to a method of increasing a population of cells in S phase in a tissue of a mammal, comprising contacting the tissue with an inhibitor (one or more) of NO, such as an inhibitor of NOS. The invention also pertains to a method of regenerating tissue in an adult mammal comprising contacting a selected tissue (e.g., blood, skin, bone and digestive epithelium), or precursor cells of the selected tissue, with an inhibitor (one or more) of NO, thereby inhibiting differentiation and inducing proliferation of cells of the tissue.

RELATED APPLICATION(S)

This application is a continuation of U.S. application Ser. No.09/315,929, filed May 20, 1999, which is a continuation-in-part of U.S.application Ser. No. 08/969,475, filed Nov. 13, 1997, which claims thebenefit of U.S. Provisional application No. 60/030,690, filed Nov. 13,1996, and the benefit of U.S. Provisional application No. 60/045,411,filed May 2, 1997. The entire teachings of the above application(s) areincorporated herein by reference.

GOVERNMENT SUPPORT

Work described herein was supported by Grant No. 5ROINS32764 from theNational Institutes of Health. The United States Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Organ development requires a tightly controlled program of cellproliferation followed by growth arrest and differentiation and, often,programmed cell death. The balance between the number of cell divisionsand the extent of subsequent programmed cell death determines the finalsize of an organ (reviewed by Bryant and Simpson, Quart. Rev. of Biol.,59:387-415 (1984); Raft, Nature, 356:397-400 (1992)). Although much ofthe cellular machinery that determines the timing of onset and cessationof cell division per se is well understood (reviewed by Hunter andPines, Cell, 79:573-582 (1994); Morgan, Nature, 374:131-134 (1995);Weinberg, Cell, 81:323-330 (1995)), little is known about the signalsthat cause discrete groups of cells and organs to terminate growth atthe appropriate cell number and size. A better understanding of thesignals involved provides possible targets for manipulating the cellularmachinery resulting in therapeutic benefits for a number of conditions.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that nitric oxide (NO)is an important growth regulator in an intact developing organism. Inparticular, the present invention relates to a method of increasing in amammal a population of hematopoietic cells (e.g., hematopoietic stemcells), including precursors to myeloid, lymphoid and erythroid cells,which are capable of undergoing normal hematopoiesis, differentiationand maturation in hematopoietic tissue, wherein the hematopoietic tissueis contacted with at least one inhibitor of NO, such as one or moreinhibitors of nitric oxide synthase (NOS), thereby producinghematopoietic tissue having an increased population of hematopoieticstem cells which are capable of undergoing normal hematopoiesis,differentiation and maturation. In one embodiment, the present inventionrelates to a method of increasing in a mammal a population ofhematopoietic stem cells which are capable of undergoing normalhematopoiesis, differentiation and maturation in hematopoietic tissue,comprising contacting the hematopoietic tissue with two inhibitors ofnitric oxide synthase, thereby producing hematopoietic tissue having anincreased population of hematopoietic stem cells which are capable ofundergoing normal hematopoiesis, differentiation and maturation. Themethod can be carried out in vivo or ex vivo. In addition, the methodcan be used to prevent differentiation of erythroid cells and/or myeloidcells in the mammal. The method can further comprise contacting thehematopoietic tissue with at least one agent (e.g., a hematopoieticgrowth factor) which induces differentiation of a selected hematopoieticstem cell population.

The present invention also relates to a method for treating a mammal toincrease a population of hematopoietic stem cells which are capable ofundergoing normal hematopoiesis, differentiation and maturation inhematopoietic tissue of the mammal. In the method, the hematopoietictissue of the mammal is contacted with at least one inhibitor of NOS,thereby producing hematopoietic tissue having an increased population ofhematopoietic stem cells which are capable of undergoing normalhematopoiesis, differentiation and maturation. In one embodiment, thepresent invention relates to a method for treating a mammal to increasea population of hematopoietic stem cells which are capable of undergoingnormal hematopoiesis, differentiation and maturation in hematopoietictissue of the mammal, comprising contacting the hematopoietic tissue ofthe mammal with two inhibitors of nitric oxide synthase, therebyproducing hematopoietic tissue having an increased population ofhematopoietic stem cells which are capable of undergoing normalhematopoiesis, differentiation and maturation. The method can furthercomprise contacting the hematopoietic tissue with at least one agentwhich induces differentiation of a selected hematopoietic stem cellpopulation.

In one embodiment of the method for treating a mammal to increase apopulation of hematopoietic stem cells which are capable of undergoingnormal hematopoiesis, differentiation and maturation in hematopoietictissue of the mammal, hematopoietic tissue which is to be transplantedis obtained, wherein the hematopoietic tissue to be transplanted can beobtained from the mammal being treated (autologous transplantation) orfrom another mammal (heterologous transplantation). The hematopoietictissue to be transplanted is contacted with at least one inhibitor ofNOS. The hematopoietic tissue which is to be transplanted istransplanted into the mammal being treated, thereby providing the mammalwith hematopoietic tissue having an increased population ofhematopoietic stem cells which are capable of undergoing normalhematopoiesis, differentiation and maturation. In one embodiment, twoNOS inhibitors are used. The method can further comprise treating themammal with an inhibitor(s) of NOS before or after transplanting thehematopoietic tissue. Alternatively, the method can further comprisetreating the mammal with an enhancer (one or more) of NOS before orafter transplanting the hematopoietic tissue.

The present invention also relates to a method of increasing apopulation of progenitor blood cells (e.g., red blood cells, white bloodcells) which are capable of undergoing normal hematopoiesis,differentiation and maturation comprising contacting progenitor cells(stem cells) of blood with at least one inhibitor of NO (e.g., aninhibitor of NOS). In one embodiment, the progenitor blood is contactedwith two inhibitors of NOS.

The present invention also relates to a method of increasing apopulation of dividing cells in a tissue of a mammal comprisingcontacting the cells with at least one inhibitor of nitric oxide. In oneembodiment, the present invention also relates to a method of increasinga population of cells in S phase in a tissue of a mammal, comprisingcontacting the tissue with an inhibitor of NO, such as an inhibitor ofNOS. In one embodiment, the method results in an increase in the size ofan organ in which the tissue is occurs. Furthermore, as described hereinthe cells in S phase can be used in gene therapy.

The present invention also relates to a method of decreasing apopulation of cells in S phase in a tissue of a mammal and inducingdifferentiation of the cells, comprising contacting the tissue with anenhancer(s) of NO, such as an enhancer of NOS. In one embodiment, themethod results in a decrease in the size of an organ with which thetissue is associated.

The present invention also relates to a method of coordinatingdevelopmental decisions of a cell type in a mammal, comprisingintroducing NO into the cell type or a precursor of the cell type,thereby inhibiting proliferation of the cell type or a precursor of thecell type and inducing differentiation of the cell type or a precursorof the cell type.

A method of inducing differentiation in a mammalian cell populationcomprising contacting the cell population with NO or a NO enhancer isalso encompassed by the present invention.

The invention also pertains to a method of regenerating tissue in anadult mammal comprising contacting a selected tissue (e.g., blood, skin,bone and digestive epithelium), or precursor cells of the selectedtissue, with at least one inhibitor of NO, thereby inhibitingdifferentiation and inducing proliferation of cells of the tissue, thencontacting the selected tissue with a compound (e.g., nitric oxide, agrowth factor or a combination of both) which inhibits proliferation andinduced differentiation. In one embodiment, the method involvesrepopulating an organ or tissue (e.g., muscle or nerve fiber) comprisedof normally nondividing cells by contacting a selected organ or tissue,or precursor cells of the selected organ or tissue, with an inhibitor ofNO, thereby inhibiting differentiation and inducing proliferation ofcells of the organ or tissue, then contacting the selected organ ortissue with a compound which inhibits proliferation and induceddifferentiation.

The invention also encompasses a method of producing a subpopulation ofhematopoietic cells. In the method, hematopoietic tissue is contactedwith at least one inhibitor of NOS, thereby producing hematopoietictissue having an increased population of hematopoietic stem cells whichare capable of undergoing normal hematopoiesis, differentiation andmaturation; and at least one agent (e.g., a hematopoietic growth factor)selected to induce specific differentiation of the hematopoietic stemcell population, thereby producing a subpopulation of hematopoieticcells. In a particular embodiment, the hematopoietic tissue is contactedwith two inhibitors of NOS.

Identification of NO as an important growth regulator in an organismprovides for various therapeutic applications in humans and othermammals.

DETAILED DESCRIPTION OF THE INVENTION

Results of the work described herein have shown that a transcellularmessenger (nitric oxide (NO)) plays a critical role in tissuedifferentiation and organism development. NO regulates the balancebetween cell proliferation and cell differentiation in the intactdeveloping organism. Increased production of NO permits cessation ofcell division and subsequent differentiation of cell in a tissue,whereas removal of the NO-mediated growth arrest promotes cell division.

Accordingly, the present invention relates to a method of increasing ina mammal a population of hematopoietic cells (e.g., hematopoietic stemcells), including precursors to myeloid, lymphoid and erythroid cells,which are capable of undergoing normal hematopoiesis, differentiationand maturation in hematopoietic tissue, by contacting the hematopoietictissue with at least one inhibitor (one or more) of NO, such as aninhibitor of NOS. As defined herein “hematopoietic tissue” is tissueinvolved in hematopoiesis. e.g., bone marrow, peripheral blood,umbilical cord vein blood, fetal liver, and long term hematopoietic cellculture.

The present invention includes a method for treating a mammal toincrease a population of hematopoietic stem cells which are capable ofundergoing normal hematopoiesis, differentiation and maturation inhematopoietic tissue of the mammal, in which the hematopoietic tissue ofthe mammal is contacted with at least one inhibitor of NOS. Theinvention also pertains to a method of producing a subpopulation ofhematopoietic cells by contacting hematopoietic tissue with at least oneinhibitor of NOS, thereby producing hematopoietic tissue having anincreased population of hematopoietic stem cells which are capable ofundergoing normal hematopoiesis, differentiation and maturation; and atleast one agent selected to induce specific differentiation of thehematopoietic stem cell population, thereby producing a subpopulation ofhematopoietic cells. In a particular embodiment, two inhibitors of NO,such as two inhibitors of NOS, are used in the methods. For example, acombination of L-NAME and ETU can be contacted with the hematopoietictissue to increase a population of hematopoietic stem cells in a mammal,to treat a mammal to increase a population of hematopoietic stem cellsin hematopoietic tissue in a mammal or to produce a subpopulation ofhematopoietic cells.

The present invention also relates to a method of increasing apopulation of progenitor blood cells comprising contacting progenitorcells of blood with at least one inhibitor (one or more) of NO (e.g.,inhibitor of NOS). In one embodiment, the present invention relates to amethod of increasing a population of progenitor blood cells comprisingcontacting progenitor cells of blood with two inhibitors of NOS. Thesources of progenitor cells of blood include, for example, bone marrow,peripheral blood, umbilical cord vein blood, fetal liver, and long termhematopoietic cell culture. Using the method of the present inventionred blood cells and white blood cells (e.g., granulocytes (neutrophils,basophils, eosinophils), monocytes, lymphocytes) can be increased.

The present invention also relates to a method of increasing apopulation of dividing cells in a tissue of a mammal comprisingcontacting the cells with at least one inhibitor of NO. In oneembodiment, the present invention can also be used to increase apopulation of cells (targeted cells) in S phase in a tissue of a mammalrelative to a similar tissue in an untreated mammal, by contacting thetissue with at least one inhibitor of NO, such as an inhibitor of NOS.In one embodiment, the method results in an increase in the size of anorgan with which the tissue is associated. Conversely, the presentinvention can also be used to decrease a population of cells in S phasein a tissue of a mammal and inducing differentiation of the cells,comprising contacting the tissue with at least one enhancer of NO, suchas an enhancer of NOS. In one embodiment, the method results in adecrease in the size of an organ with which the tissue is associated.Furthermore, as described herein the cells in S phase can be used ingene therapy.

The present invention also relates to a method of coordinatingdevelopmental decisions of a cell type in a mammal, comprisingintroducing NO into the cell type or a precursor of the cell type,thereby inhibiting proliferation of the cell type or a precursor of thecell type and inducing differentiation of the cell type or a precursorof the cell type. A method of inducing differentiation in a mammaliancell population comprising contacting the cell population with NO or aNO enhancer is also encompassed by the present invention.

The invention also pertains to a method of regenerating tissue in anadult mammal. The method comprises contacting a selected tissue with atleast one inhibitor of NO, thereby inhibiting differentiation andinducing proliferation of cells of the tissue, then contacting theselected tissue with a compound which inhibits proliferation and inducesdifferentiation of the proliferated cells to cells characteristic of thetissue. In one embodiment, the method involves repopulating an organ ortissue (e.g., muscle or nerve fiber) having normally nondividing cellscomprising contacting a selected organ or tissue with an inhibitor(s) ofNO, thereby inhibiting differentiation and inducing proliferation ofcells of the organ or tissue, then contacting the selected organ ortissue with a compound which inhibits proliferation and inducesdifferentiation of the proliferated cells to cells characteristic of theorgan or tissue. Compounds which inhibit proliferation and inducedifferentiation include NO, an enhancer of NO and a growth factor. Oneor more these compounds can be used to inhibit proliferation and inducedifferentiation.

Tissue which can be regenerated using the methods described hereininclude blood, skin, bone and digestive epithelium, nerve fiber, muscle,cartilage, fat or adipose tissue, bone marrow stroma and tendons.

The methods described herein can further comprise the step of contactingthe hematopoietic tissue target cells (e.g., bone marrow) with at leastone agent which induces differentiation of a selected hematopoietic stemcell population to a particular cell type (e.g., erythrocytes,macrophages, lymphocytes, neutrophils and platelets). For example, inthe embodiment wherein a mammal is treated to increase a population ofhematopoietic stem cells in the hematopoietic tissue of the mammal bycontacting the hematopoietic tissue of the mammal with an inhibitor ofNOS, the increased population of hematopoietic tissue can be contactedwith an agent, such as a hematopoietic growth factor, which will causeor promote differentiation of the cells of a particular cell type.Agents (e.g., such as hemopoietic growth factors) which can be used inthe methods of the present invention to induce differentiation of theincreased or expanded number of cells produced by contacting cells witha NOS inhibitor include, for example, erythropoietin, G-CSF, GM-CSF andinterleukins such as IL-1, IL-2, IL-3 and IL-6. Alternatively, themethods described herein can further comprise the step of contacting thehematopoietic tissue with at least one agent which further induces ormaintains proliferation of the selected hematopoietic stem cellpopulation to a particular cell type (e.g., erythrocytes, macrophages,lymphocytes, neutrophils and platelets).

Inhibitors of NO for use in the present invention include, for example,NO scavengers such as2-phenyl-4,4,5,5-tetraethylimidazoline-1-oxyl-3-oxide (PTIO),2-(4-carboxyphenyl)-4,4,5,5-tetraethylimidazoline-1-oxyl-3-oxide(Carboxy-PTIO) and N-methyl-D-glucamine dithiocarbamate (MGD); and NOSinhibitors such as N-nitro-L-arginine methyl-ester (L-NAME),N-monomethyl-L-arginine (L-NMMA), 2-ethyl-2-thiopseudourea (ETU,),2-methylisothiourea (SMT), 7-nitroindazole, aminoguanidine hemisulfateand diphenyleneiodonium (DPI).

In the methods of the present invention, at least one inhibitor of NO(e.g., NOS inhibitors) can be used. When more than one inhibitor is usedin the methods of the present invention, the inhibitors can be the sameor different. In a particular embodiment, two inhibitors of NO, such astwo inhibitors of NOS (e.g., L-NAME and ETU), are used in the methods ofthe present invention.

Furthermore, in the methods of the present invention, the NOinhibitor(s) can be administered in a single dose or in multiple doses.The multiple doses can be administered in a day or over a period of days(e.g., a period of about 2 days to a period of about 15 days). Forexample, the NO inhibitor(s) can be administered over 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 days. In one embodiment, a mixture oftwo NO inhibitors (e.g., L-NAME and ETlU) are administered to the mammalor contacted with the cells twice a day for 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 days. In a particular embodiment, L-NAME and ETU areadministered to the mammal or contacted with the cells twice a day for 9days.

In the methods of the present invention, one or more enhancers of NO canbe used. Enhancers of NO include, for example, NOS enhancers, and NOdonors such as sodium nitroprusside (SNP),S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (SNOG,GSNO), diethylamine NONOate (DEA/NO), DETA/NO (NOC-18),3-morpholinosydnonimine (SIN-1) and spermine NONOate (Sper/NO).

NO is a diffusible multifunctional second messenger that has beenimplicated in numerous physiological functions in mammals, ranging fromdilation of blood vessels to immune response and potentiation ofsynaptic transmission (Bredt and Snyder, Annu. Rev. Biochem., 63:175-195(1994); Nathan and Xie, Cell, 78:915-918 (1994); Garthwaite and Boulton,Annu. Rev. Physiol., 57:683-706(1995)). NO is produced from arginine byNOS in almost all cell types. A group of three chromosomal genes, givingrise to numerous isoforms of NOS, have been cloned from mammalian cells(Knowles and Moncada, Biochem. J, 298:249-259 (1994); Wang and Marsden,Adv. Pharmacol., 34:71-90 (1995) ), and recently a Drosophila NOS gene,whose coding structure resembles the gene for the mammalian neuronalisoform, has been isolated (Regulski and Tully, Proc. Natl. Acad. Sci.USA, 92:9072-9076 (1995)).

Cell division and subsequent programmed cell death in imaginal discs ofDrosophila larvae determine the final size of organs and structures ofthe adult fly. Results described herein show that NO is involved incontrolling the size of body structures during Drosophila development.These results demonstrate that NOS is expressed at high levels indeveloping imaginal discs. Inhibition of NOS in larvae causeshypertrophy of organs and their segments in adult flies, whereas ectopicexpression of NOS in larvae has the opposite effect. Blocking apoptosisin eye imaginal discs unmasks surplus cell proliferation and results inan increase in the number of ommatidia and component cells of individualorumatidia. These results demonstrate the activity of NO as anantiproliferative agent during Drosophila development, controlling thebalance between cell proliferation and cell differentiation. Moreover,results shown here demonstrate that NO acts as a crucial regulator ofhematopoiesis after bone marrow (BM) transplantation. NO regulates thematuration of both the erythroid and myeloid lineages. These datademonstrate that manipulations of NOS activity and NO levels duringhematopoiesis can be used to alter (enhance or reduce) blood cellproduction. This is useful for preventive and therapeutic intervention.

During Drosophila development, the structure, size, and shape of most ofthe organs of the adult fly are determined in the imaginal structures ofthe larvae (Cohen, Imaginal disc development, in The Development ofDrosophila melanogaster, M. Bate and A. Martinez-Afias, eds. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), 747-841(1993); Fristrom and Fristrom, The metamorphic development of the adultepidermis, in The Development of Drosophila melanogaster, M. Bate and A.Martinez-Afias, eds. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.), 843-897 (1993)). Imaginal discs, specialized groups ofundifferentiated epithelial cells that are recruited duringembryogenesis, are formed in the first larval instar as integuments ofthe larval epidermis. Disc cells divide rapidly throughout the larvaldevelopment and cease proliferating at the end of the third instarperiod.

In leg, wing, and haltere discs, progression through the cell cyclestops in G2 phase 3-4 hours before puparium formation. It resumes 15-18hours later (12-14 hours after pupariation) and then stops again in adefined spatial pattern after 12-14 hours (10-14 hours of pupaldevelopment) (Fain and Stevens, Dev. Biol., 92:247-258 (1982); Gravesand Schubiger, Dev. Biol., 93:104-110 (1982); Schubiger and Palka, Dev.Biol., 123:145-153 (1987)). Although most of the dividing cells in thelate larvae and in the early pupae are already committed to their adultfate, they do not develop a fully differentiated phenotype until growtharrest is firmly established. Thus, cell proliferation is temporallyseparated from cell differentiation, which takes place later duringmetamorphosis. Experiments with transplanted imaginal discs suggest thatcessation of cell proliferation in these structures is controlled bymechanisms that, while intrinsic to the disc, are not completelycell-autonomous (Bryant and Schmidt, J. Cell Sci., Suppl. 13:169-189(1990); Cohen, Imaginal disc development, in The Development ofDrosophila melanogaster, M. Bate and A. Martinez-Afias, eds. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), 747-841(1993)). The signaling pathways that control coordinated temporarygrowth arrest in larvae and pupae and subsequent terminal growth arrestin pupae and adults are not known, but they probably involveintercellular and intracellular second messenger molecules which havenot yet been identified.

Transformation of imaginal precursors in adult structures during flymetamorphosis involves transition from cell proliferation to celldifferentiation. Cessation of cell division is a necessary, although notsufficient, condition for cell differentiation to proceed. A temporarycytostasis occurs at the end of the larval period, and permanent arrestof cell division occurs during pupal development. NO, a diffusiblemessenger molecule, is capable of efficiently blocking cell division.Induction of NOS initiates a switch to growth arrest prior todifferentiation of cultured neuronal cells (Peunova and Enikolopov,Nature, 375:68-73 (1995)). Thus, NOS can act as a permissive factor,making the further development of the fully differentiated phenotypepossible. Results described herein show that NOS acts as anantiproliferative agent during normal Drosophila development, indicatingthat NO is an important growth regulator in the intact developingorganism.

Throughout larval development, there is a gradual and spatially-specificaccumulation of NADPH-diaphorase activity in developing imaginal discs,reflecting an increase in overall NOS content. At the time temporarycytostasis is being established in imaginal discs, NADPH-diaphorasestaining becomes particularly intense, and it gradually decreases duringprepupal and pupal development. Besides the imaginal discs, otherstructures with intense NADPH-diaphorase staining include imaginalrings, histoblasts and the brain. These structures undergo radicalchanges during metamorphosis before giving rise to adult organs. Theirdevelopment includes periods of rapid cell division alternating withperiods of cytostasis, and thus must employ mechanisms for coordinatedcessation of DNA synthesis and cell division in a spatially definedpattern. Since NO can prevent cell division and can diff-use and actwithin a limited volume, the ability of NO to act to induce coordinatedgrowth arrest during Drosophila development was considered. Indeed, ifNO actively exerts its antiproliferative activity during the developmentof imaginal discs, then inhibition of NOS before the temporarycytostasis is established at the end of the larval period could lead tothe reversal of the arrest of cell division and induce additionaldivisions, which in turn could lead to increased size of structures ofthe body of the adult fly. Conversely, excessive or ectopic-productionof NO in larvae could cause premature cessation of cell division andlead to a reduction in the size of the structures in the adults.

Both predictions were confirmed in experiments described herein, inwhich NOS activity was manipulated in the developing fly. NOS inhibitionin larvae caused an increase in the number of cells in some parts of theadult body and an increase in their size, whereas ectopic expression ofthe NOS transgene during development caused a decrease in the number ofcells in some structures in the adult and a decrease in their size,probably by partial fusion and reduction. In the developing leg, thesegments that were most often affected when NOS activity was inhibitedand the segments that were most often affected when the activity wasectopically induced were nonoverlapping and complementary. Mostimportantly, their distribution matched the distribution of NOS in theimaginal discs, thereby supporting the hypothesis that NO plays acausative role in growth arrest in normal development.

The antiproliferative properties of NO suggest that NOS acts indevelopment through its influence on DNA synthesis and cell division.The results described herein with BrdU incorporation in leg discs withelevated and diminished production of NO conoborate this position andsuggest a direct link between synthesis of NO, number of S-phase cells,and the final size of the organ. In accordance with this idea, in manyinstances no BrdU incorporation was observed in regions highly enrichedin NOS. The mechanisms for the NO-mediated arrest of the cell cycle(both temporary and terminal) are not clear, but they likely involve theconventional cellular machinery for growth arrest, e.g., cellcycle-dependent kinases and their inhibitors. Consistent with this,changes in expression of these proteins were observed when culturedcells were treated with NO. An intriguing feature of imaginal disc cellsis that they stop dividing and accumulate in G2 phase in the late thirdinstar, preceding the period of temporary cytostasis (Fain and Stevens,Dev. Biol., 92:247-258 (1982); Graves and Schubiger, Dev. Biol.,93:104110 (1982); Schubiger and Palka, Dev. Biol., 123:145-153 (1987)).This parallels a tendency of NO-treated (Peunova and Enikolopov, Nature,375:68-73 (1995)) and NGF-treated (Buchkovich and Ziff, Mol. Biol. Cell,5:225-241 (1994)) PC12 cells to accumulate in G2 phase. Interestingly,imaginal discs are released from the G2 block and reenter S phase 12-15hours after pupariation, at the time when diaphorase staining isdiminished to low levels in adult flies. These correlations betweenimaginal discs cells and NO-treated cells support the idea that NO canbe a major inducer of cytostasis in the cells of imaginal discs in theprepupal stage.

The final number of cells in an organ or a segment is determined both bycell multiplication and cell death, which the forming structures of thefly undergo as a normal step in development (especially at the latestages of pupal development). Results described herein indicate that thechanges in the size of the leg segments after manipulation of NOSactivity correlated directly with the changes in DNA synthesis and thenumber of dividing cells. Furthermore, no significant changes inapoptosis were detected in the larval and prepupal leg discs afterinhibition or ectopic expression of NOS, compared with the controldiscs, when cell death was monitored by acridine-orange staining or bythe TUNEL assay. This suggests that it is cell multiplication, ratherthan changes in programmed cell death that leads to the changes in thesize of the appendage.

On the other hand, apoptotic death may conceal excessive cellproliferation in other developing organs. The effect of the absence ofprogrammed cell death on potential excessive cell proliferation was alsoassessed. Transgenic flies were used in which programmed cell death inthe developing eye was suppressed by recombinant p35, an inhibitor ofapoptosis, to reveal excessive proliferation after NOS inhibition. Underthese circumstances, several cell types and structures areoverrepresented, the most noticeable change being an overall increase ofthe size of the eye due to the increased number of ommatidia. Inaddition, other cell types (e.g., secondary and tertiary pigment cells,cone cells, and cells of the bristles) proliferated after NOS inhibitionto levels higher that those achieved by blocking apoptosis by p35 (Hayet al., Dev., 120:2121-2129 (1994)). These data demonstrate that theremoval of suppressive influence of NO leads to an increase in the sizeof the adult organ, unless this effect is masked by programmed celldeath, and indicate that final cell number in the adult organ is underdual control by both cell proliferation and programmed cell death.Furthermore, these data provide independent support for the hypothesisthat NO directly regulates cell number during development.

After inhibition of NOS with either of two structurally unrelatedcompounds, excessive growth was observed in most of the structures ofthe adult flies that derive from imaginal discs and histoblasts, tovarying extents for different organs. The most obvious changes wereobserved in the segments of the legs whose primordia showed the highestlevels of NOS. There did not appear to be any substantial number ofinstances in which a duplication of a larger structure (for example,segments of the legs or wings) occurred. This indicates that extraproliferation of cells under the influence of NOS inhibitors occursafter the developmental fate is determined for most of the cells in theimaginal discs. This suggests that in most cases NO may be moreimportant for the induction of growth arrest and subsequentdifferentiation of already committed cells than for the developmentalcommitment and establishment of the cell identity in the embryo orlarvae.

Only some of the axes of the developing structures were affected bymanipulations of the NOS activity. For instance, in developing legs onlythe anteroposterior and the dorsoventral axes, but not the proximodistalaxis, were affected by inhibition of NO production. In contrast, whenNOS was ectopically expressed, only the proximodistal axis was affected.These results suggest that a gradient of NO may be involved in theprocess of establishing the polarity of the axes of the developingorgan.

Thus these results demonstrate that inhibition of NOS in larvae leads toenlargement of organs in adults and, conversely, that ectopic expressionof NOS in larvae leads to a reduction in the size of organs in adults.Also, the distribution of affected segments in the adult leg correspondsto the distribution of NOS in the larvae, and the changes in segmentsize can be directly correlated to changes in DNA synthesis in imaginaldiscs after manipulations of NOS activity. The increased cellproliferation that occurs in response to NOS inhibition is masked insome structures by programmed cell death, and it can be revealed bysuppressing apoptosis. Taken together, these results demonstrate thatactivation of NOS is a crucial step in Drosophila development. Theyconfirm that NO acts as an antiproliferative agent during celldifferentiation and organism development and controls the cell number inan intact developing organism.

NOS expression can be induced to high levels in a large number oftissues and cell types by appropriate stimulation (Bredt and Snyder,Annu. Rev. Biochem., 63:175-195 (1994); Forstermann et al., Adv.Pharmacol., 34:171186 (1995)). In most cases, the pattern of NOSdistribution in a developing organism differs strongly from thedistribution in the adult organism. Furthermore, transient elevation ofNOS expression in a given tissue often coincides with the cessation ofdivision of committed precursor cells. The developing mammalian brainprovides an especially apt demonstration of this (Bredt and Snyder,Neuron, 13:301-313 (1994); Blottner et al., Histochem. J, 27:785-811(1995)). A strong elevation of NOS activity in the developing cerebralcortical plate and hippocampus at days 15-19 of prenatal developmentcorrelates with the timecourse of cessation of precursor cellsproliferation, tight growth arrest, and cell differentiation; notably,NOS activity goes down after the proliferation of committed neuronalprecursors is completed. NOS levels are also transiently increased indeveloping lungs, bones, blood vessels, and nervous system (Blottmer etal., Histochem. J., 27:785-811 (1995); Collin-Osdoby et al., J. CellBiochem., 57:399-408 (1995); Cramer et al., J. Comp. Neurol.,353:306-316 (1995); Shaul, Adv. Pediatr., 42:367-414 (1995); Wetts etal., Dev. Dyn., 202:215-228 (1995)). Elsewhere, NOS activity is greatlyelevated in regenerating tissues when cessation of cell division iscrucial for prevention of the unregulated growth (Roscams et al.,Neuron, 13:289-2Y9 (1994); Blottner et al., Histochem. J, 27:785-811(1995); Decker and Obolenskaya, J. Gastroenterol. Hepatol., 10 Suppl1:2-7 (1995); Hortelano et al., Hepat. 21:776-786 (1995)). In all thesecases, a transient elevation of NOS activity might trigger a switch fromproliferation to growth arrest and differentiation, thus contributing tothe proper morphogenesis of the tissue and the organ.

Results described herein support the position that production of NO isrequired during embryonic development and during tissue regeneration inthe adult organism for the proper control of cell proliferation. Theantiproliferative properties of NO are particularly important insituations in which terminal differentiation of committed cells istemporally separated from cell proliferation and is strictly dependenton cessation of cell division. Given the multiplicity of the NOSisoforms and their overlapping tissue distribution, it is conceivablethat any group of cells in the embryo and fetus can be exposed to NOaction. Furthermore, recent data showing that NO can be transferredwithin the organism by hemoglobin (Jia et al., Nature, 380:221226(1996)) raise the possibility that a developing mammalian embryo can bealso supplied with NO exogenously by the mother.

NO is a readily diffusible molecule, and it may therefore exert itsantiproliferative properties not only in the cell that produces it butin the neighboring cells as well (Gally et al., Proc. Natl. Acad. Sci.USA, 87:3547-3551 (1990)). This property is important when one considersmechanisms for the coordinated development of a group of neighboringcells committed to form a particular structure. These cells have togenerate an intrinsic signal that tells them to stop dividing in acoordinated fashion after they have reached a certain number. Thiscooperation and coordination is achieved in many instances by tightlycontrolled paracrine regulation, which involves signaling betweenadjacent cells via gap junctions or secreted proteins. Results describedherein show that yet another way of coordinating developmental decisionsin groups of cells is by diffusible antiproliferative second messengermolecules, which can spread without a need for surface receptors orspecialized systems for secretion and exert their influence within alimited domain. An efficient source of readily diffusible molecules mayinduce synchronized changes in the adjacent cells within a limitedvolume of a tissue. Moreover, several adjacent cells producing easilydiffusible antiproliferative messenger molecules may share the totalpool of these molecules produced by the neighbors as well as bythemselves. If a particular threshold level of a signal is needed toinitiate a signaling chain that eventually leads to growth arrest, thenthe cells in this group could stop dividing when a certain number ofcells and, therefore, a certain local concentration of messengermolecules, is reached. In this way, by organizing groups of cells infunctional clusters and coordinating their decisions on proliferationand differentiation, No instruct the developing structures to terminatetheir growth when they attain the appropriate size and shape, and, thus,participate in tissue and organ morphogenesis.

As also described herein, the role of NO in hematopoiesis was examined.To demonstrate the presence of NOS in the bone marrow (BM) cells, BMfrom adult mice was tested for the NDPH-diaphorase activity of NOS(which reflects the distribution of the total enzyme activity in atissue). It was found that BM contains a substantial proportion of cells(up to 12%) with strong diaphorase staining. The morphology of theNADPH-diaphorase cells suggests that they are largely of thegranulocyte-macrophage lineage at different stages of differentiation.This is in accordance with numerous data showing that NOS is present inthe cells of the myeloid lineage, and can be induced to high levels byappropriate stimulation.

A mouse model of syngeneic BM transfer was used to evaluate the role ofNO in hematopoiesis. Mice were irradiated to inhibit hematopoiesis inthe recipient animal, BM was transplanted from syngeneic animals, andthe animals were treated with specific NOS inhibitors. This procedurepermits the proliferation, differentiation and survival of only thetransplanted cells. To study the changes in hematopoiesis introduced byNOS inhibitors, the colonies in the spleen were monitored to test thedifferentiation of erythroid cells, and the formation of colonies on themembranes placed in the peritoneal cavity of the recipients weremonitored to test the differentiation of cells of thegranulocyte-macrophage lineage. The role of NO on hematopoiesis wastested by injecting the animals with the specific and structurallyunrelated NOS inhibitors L-nitroarginine methyl ester (L-NAME), and2-ethyl-2-thiopseudourea (ETU). The inactive enantiomer D-NAME was usedas a control. Animals were sacrificed and the number and composition ofcolonies in the spleen (reflecting the cells which have undergoneerythroid differentiation) and colonies on the membranes (reflecting thecells that have undergone myeloid differentiation) were studied.

Taken together, the results of these studies indicate that NO modulateshematopoiesis after BM transplantation. This confirms the role of NO asa major regulatory factor in the organism controlling the balancebetween proliferation and differentiation. This also shows thatmanipulation of NO levels may be used for therapeutic intervention toincrease the number of undifferentiated hematopoietic cells after BMtransplantation; change the ratio of cells undergoing erythioid ormyeloid differentiation; and interfere or suppress graft-versus-hostdisease, which is a major cause of mortality in patients undergoing BMtransplantation.

Most of the tissues and organs in the adult organism are constantlyundergoing regeneration and renovation, going through phases of rapidproliferation, determination, growth arrest, differentiation, and often,programmed cell death. Many human diseases are caused by improper orincomplete differentiation steps, resulting in the loss of function of aparticular tissue or organ. This suggests that these diseases can betreated, and, furthermore, proper function of the affected tissues andorgans can be restored by targeting and manipulating cell and tissuedifferentiation.

This work described herein, demonstrating the role of NO in cellproliferation and differentiation in an organism, provides for varioustherapeutic applications in humans and other mammals. In particular,this NO-based approach can be focused on renewable and regeneratingtissues, such as blood, bone, skin, and digestive epithelium.Additionally, a similar strategy can be used to repopulate organs withnormally nondividing cells such as muscle and nerve cells.

The work described herein can also be used to enhance gene therapymethods. For example, NOS can be used to drive a population of cellsinto the S phase wherein the cells are replicating. As known in the art,replicating cells are more responsive to gene therapy methods (e.g.,introduction of genes via live vectors) than non-replicating cells.Thus, the present invention provides for a method of converting cellsinto a state which renders the cells more receptive to gene therapymethods, wherein the cells are contacted with a NO inhibitor (e.g., NOSinhibitor). Conversely, the present invention provides for a method ofconverting cells into a state which renders the cells resistant to genetherapy methods. That is, the present invention provides for a method ofconverting cells into a state which renders the cells more resistant togene therapy methods, wherein the cells are contacted with NO and/or aNO enhancer (e.g., NOS enhancer).

The results of work described herein support the ability of NO to act asa crucial regulator of hematopoiesis after bone marrow transplantation(BMT). NO regulates maturation of both erythroid and myeloid celllineages. By interfering with NO production in the recipient animalafter BMT, the number of undifferentiated stem and blast cells which arethen capable of further differentiation along the erythroid or myeloidlineages can be dramatically increased. The blast cells enrichmentreaches 80-fold for the myeloid lineage, and 20-fold for the erythroidlineage. The data described herein demonstrates that manipulations ofNOS activity and NO levels during hematopoiesis can be used fortherapeutic purposes to influence self renewal and differentiation ofhematopoietic stem cells, and to replace damaged or defective cells.Areas of application include enhancement of blood cell and myeloid cellformation following high dose chemotherapy in cancer treatment; improvedengraftment following bone marrow or stem cell transplantations, andgene therapy; stem cell therapy by amplifying the undifferentiated cellsof erythroid and myeloid lineages and applying appropriate factors toinduce terminal differentiation; and regulation of formation of variousblood cell components for treating hematological and autoimmunedisorders.

The data also shows that changing the levels of NO production interfereswith osteoblast and chondrocyte differentiation. These results show thatmanipulation of NO production can regulate growth and differentiation ofosteoblasts, chondrocytes, or mesenchymal stem cells. This can be usedfor amplification and further differentiation of cells in the injuredtissue, or for cell implants (in combination with biocompatiblecarriers, if necessary). Thus, an NO-based approach can be used forregeneration therapy of the damaged tissue, post injury repair, agerelated diseases such as osteoporosis and osteoarthritis; and forreconstituting marrow stroma following high dose cancer chemotherapy.

In addition, the data shows that changing the levels of NO productioninterferes with keratinocyte differentiation. The results describedherein demonstrate that regulation of NO production can be used whenincreased proliferation and subsequent differentiation of skin tissue isrequired (e.g., during burns and wound healing). Furthermore, NO can beused to control disorders caused by hyperproliferation of keratinocytesduring psoriasis. Yet another potential application is to use NO-basedpreparations as exfoliant agents in cosmetic therapy.

NO has been shown to act as a regulator of cell differentiation inneuronal cells. It has been demonstrated that NO regulates braindevelopment in animals and contributes to controlling the size of thebrain in intact animals.

It has also been demonstrated that in certain contexts NO mediates thesurvival effects of growth factors by activating an antiapoptiticprogram and can protect neuronal cells from death. Combined, thesestudies of the role of NO in neurons suggest that NO may be used tocontrol proliferation and subsequent differentiation of nerve cells inreplacement therapy after neurodegenerative disorders caused by aging(e.g., Alzheimer's or Parkinson's), stroke, or trauma.

NO is actively produced in smooth muscle cells of the blood vessels andis subject to complex physiological regulation. These cells are highlysusceptible to suppression of DNA synthesis by NO. The very strongantiproliferative activity of NO can be used for inhibition of smoothmuscle cells proliferation and neointima formation for treatment ofrestenosis following angioplasty.

In addition, NO-based therapy has application for treatment of ailmentscharacterized by destruction of specific sets of cells. This includeshepatocyte regeneration after toxic injury of the liver, treatment ofreproductive system disorders, and administration of differentiatedpancreatic tissue for treatment of type 1 diabetes.

The methods of the present invention can be carried out in vivo or exvivo. Administration of the NO inhibitor, NO enhancer and/or agent whichinduces differentiation can performed using various delivery systemsknown in the art. The routes of administration include intradermal,transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous,oral, epidural and intranasal routes. Any other convenient route ofadministration can be used such as, for example, infusion or bolusinjection, or absorption through epithelial or mucocutaneous linings. Inaddition, the NO inhibitor, NO enhancer and/or agent which inducesdifferentiation can be administered with other components orbiologically active agents, such as adjuvants, pharmaceuticallyacceptable surfactants, excipients, carriers diluents and vehicles.Administration can be systemic or local, e.g., direct injection at thesite containing the cells to be targeted. In the embodiment, in whichthe NO inhibitor, NO enhancer and/or agent which induces differentiationare protein or peptides, they can be administered by in vivo expressionof genes or polynucleotides encoding such into a mammalian subject.Several expression systems, such as live vectors, are availablecommercially or can be reproduced according to recombinant DNAtechniques for use in the present invention.

The amount of NO inhibitor, NO enhancer and/or agent which for use inthe present invention which will be effective in the treatment of theparticular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration and the seriousness of the diseaseor disorder, and should be decided according to the judgement of thepractitioner and each patient's circumstances. For example, the amountof NO inhibitor(s) for use in the methods of the present invention canbe from about 1 mg/kg body weight to about 1000 mg/kg body weight, fromabout 5 mg/kg body weight to about 500 mg/kg body weight, and from about25 mg/kg body weight to about 100 mg/kg body weight. In one embodiment,about 25 mg/kg body weight to about 1000 mg/kg body weight L-NAME and/orabout 1 mg/kg body weight to about 100 mg/kg body weight can be used inthe methods of the present invention. In a particular embodiment, about300 mg/kg body weight L-NAME is used in combination with about 30 mg/kgbody weight ETU in the methods of the present invention.

The following Examples are offered for the purpose of illustrating thepresent invention and are not to be construed to limit the scope of thisinvention. The teachings of all references cited herein are herebyincorporated herein by reference.

EXAMPLES Example 1 Nitric Oxide Regulates Cell Proliferation DuringDrosophila Development

Drosophila Stocks.

Drosophila melanogaster Oregon R strain was used for most of theexperiments described. Transgenic GMR-P35 flies (3.5 and 2.1 alleles,Hay et al., Dev., 120:2121-2129 (1994)) were a generous gift from B. Hayand G. M. Rubin. Transgenic flies carrying mouse macrophage NOS (NOS2)gene under heat-shock promoter (hs-mNOS20(2) and hs-mNOS 15(2) alleles)were generated by P-element-mediated germline transformation. A 4100base pair NotI fragment from the plasmid CL-BS-mac-NOS containing theentire mouse macrophage NOS gene (Lowenstein et al., Proc. Natl. Acad.Sci. USA, 89:6711-6715 (1992)) was cloned into the NotI site in the Pelement vector pP(CaSpeR-hs) (Thummel and Pirrotta, DrosophilaInformation Service, 71:150 (1992)), placing it under the control of theDrosophila hsp70 promoter. The construct was coinjected into embryos(Spradling., P element-mediated transformation, in Drosophila: Apractical approach, D. B. Roberts, ed. (Oxford: IRL Press) 60-73 (1986))with the helper P element phs-II-Δ2-3 (Misra and Rio, Cell, 62:269-284(1990)). A set of two independent, homozygous transformants wereestablished. Expression of the NOS2 transgene after heat-shock treatmentof larvae and adult flies was confirmed by diaphorase staining and byprotein and RNA analysis. In control experiments, identical regimens ofheat-shock treatment of nontransformed flies did not induce anyanatomical changes per se.

Histochemistry and Electron Microscopy

NADPH-diaphorase staining was performed as described by Dawson et al.,Proc. Natl. Acad. Sci. USA, 88:7797-7801 (1991) and Hope et al., Proc.Natl. Acad. Sci. USA, 88:2811-2814 (1991), with minor modifications.Fixation-insensitive NADPH-diaphorase staining reflects activity ofvarious NOS isoforms in mammals and Drosophila. Imaginal discs weremounted in 80% glycerol and photographed in a Zeiss Axiophot microscopeunder Nomarski optics. Cobalt-sulfide staining of the pupal retinae wascarried out as described by Wolff and Ready, Dev., 113:825-839 (1991).BrdU labeling to identify cells in S phase was performed essentially asdescribed by Schubiger and Palka, Dev. Biol., 123:145-153 (1987) and byBaker and Rubin, Dev. Biol., 150:381-396 (1992), with minormodifications. Imaginal discs were removed, rinsed, and incubated inSchneider's media in 50 μg/ml solution of BrdU for 30-40 minutes at roomtemperature. They were fixed in 4% formaldehyde, treated with 1:1mixture of heptane and formaldehyde, rinsed, depurinated by 1M HCl,blocked by 1% sheep serum, and incubated with anti-BrdU antibodies(Beckton-Dickinson). After extensive washing, discs were incubated withfluoresceine-coupled anti-mouse secondary antibodies(Boehringer-Mannheim). After rinsing, individual imaginal discs weredissected away, dehydrated in ethanol, and mounted in Vectashieldmounting media (Vector Laboratories). Scanning electron microscopy wasperformed at the SUNY Stony Brook Microscopy Center essentially asdescribed by Kimmel et al., Genes Dev., 4:712-727 (1990). The number ofommatidia was determined both by analyzing series of scanning electronmicrographs and by analyzing adult heads under the blue fluorescentlight in a Zeiss Axiophot microscope.

Microinjection of Larvae

For inhibition of NOS, third instar larvae were injected withL-nitroarginine methyl ester (L-NAME), its inactive enantiomerD-nitroarginine methyl ester (D-NAME) (both from Sigma), and2-ethyl-2-thiopseudourea (ETU; Calbiochem). Chemicals were dissolved inSchneider's solution at concentrations of 0.1M for L-NAME and D-NAME and0.01M for ETU and mixed with Freund's adjuvant (Sigma) in 1:3 ratio.Amounts of 5-10 nl were microinjected in staged late third instar usinga glass needle. Timing of the injection of NOS inhibitors that gave thehighest efficiency (as determined by the changes in the phenotype of theadults) was determined in trial experiments and was found to be mostefficient when performed 5-12 hours before pupanation. This treatmentdid not affect the onset of pupanation and hatching.

Ectopic Expression of NOS

For regulated ectopic expression of NOS, larvae carrying the mouse NOS2cDNA under the control of Drosophila heat-shock promoter were treatedwith heat shock at 36° C. for 40 minutes within the first hour afterpuparium formation. For BrdU labeling experiments, third instar larvaewere treated with heat shock 5-8 hours before puparium formation.

Results

NOS is expressed in imaginal discs during larval development.

At the end of the third instar, cells of imaginal discs undergotemporary cell cycle arrest. Cytostasis is released 12-14 hours afterpupariation and is established once again (permanently) in the latepupae and the pharate adult. The ability of NO to reversibly halt celldivision and establish temporary growth arrest makes it a plausiblecandidate for mediating cytostasis in imaginal discs. To investigatethis possibility, imaginal discs of the third instar and early pupaewere examined for NOS presence. Drosophila NOS (dNOS) gene, which ispreferentially expressed in the adult head, has recently been cloned andcharacterized (Regulski and Tully, Proc. Natl. Acad. Sci. USA,92:9072-9076 (1995)). However, different NOS-related mRNA species arepresent in the embryo, larvae and adult flies. These mRNAs may beproduced by the cloned dNOS gene or by other potential Drosophila NOSgenes, making the detection of the relevant RNA species difficult.Therefore, to visualize the expression of NOS in Drosophila duringlarval development, histochemical staining for the NADPH-diaphorase(reduced nicotinamide adenine dinucleotide phosphate-diaphorase)activity of NOS was used, which reflects the distribution of the totalenzyme activity in a tissue (Dawson et al., Proc. Natl. Acad. Sci. USA,88:7797-7801 (1991); Hope et al., Proc. Natl. Acad. Sci. USA,88:2811-2814 (1991); Muller, Eur. J. Neurosci., 6:1362-1370 (1994)).

NADPH-diaphorase staining was observed in all imaginal discs, imaginalrings, histoblasts and the brain of the larvae, beginning in the thirdinstar. Staining became more intense as development proceeded, and inlate third instar larvae and early pupae, a highly specific andreproducible pattern of very intense staining was evident. In the legimaginal disc NADPH-diaphorase staining was initially seen at the verybeginning of the third instar. Staining was confined to the center ofthe disc, corresponding to the presumptive distal tip of the leg. As thediscs matured, diaphorase staining intensified, and in the late thirdinstar it nearly obliterated the distinction between individualconcentric rings of epithelial folding normally seen in axial view. Atthe end of the third instar stage, the staining of the center of thedisc (distal tip), which stained most darkly at the beginning of thethird period, was weaker in comparison with the surrounding cells. Laterin development, when the discs began to evert in the prepupae,diaphorase staining of the forming leg became less intense, and adistinct characteristic pattern of staining of individual segmentsbecame evident. At 2-4 hours after puparium formation, intenseNADPH-diaphorase staining was observed in the presumptive tibia, firstand second tarsal segments, and the proximal part of the fifth tarsalsegment of the forming leg. Staining was much weaker in the third andfourth segments, and areas of intense staining were unevenly distributedthroughout the regions of presumptive femur. Weak staining was alsopresent in the coxa and body wall. The progression of staining patternsthroughout the larval development was highly specific and reproducible.The staining of the imaginal discs corresponding to the first, secondand third pairs of legs was very similar. As with the leg imaginaldiscs, other imaginal discs, imaginal rings and histoblasts exhibitedincreasingly intense NADPH-diaphorase staining as larval developmentproceeded. Wing, eye, haltere and genital discs in the third instar haddistinct and reproducible patterns of intense staining, which graduallydecreased in a specific spatial pattern during early pupal development.

These results demonstrate that there is a gradual and specificaccumulation of NOS in developing imaginal discs, which reaches highestlevels at the time when the progression through the cell cycle slowsdown.

Synthesis of DNA is Affected by Manipulations of NOS Activity.

If NO acts as an antiproliferative agent during Drosophila developmentat stages when the cells of imaginal discs enter temporary cytostasis,then its action might directly affect DNA synthesis in the discs.Inhibition of NOS would then be expected to relieve the block andincrease the number of cells in S-phase; conversely, high levels of NOwould lead to a decrease in the number of dividing cells. To test thishypothesis and to map the extent and distribution of theantiproliferative effect of NO, DNA synthesis in larval and prepupaldiscs was monitored while the levels of NOS activity were manipulated.To inhibit NOS activity, specific NOS inhibitors were injected intodeveloping larvae. To increase the levels of NOS, expression of NOStransgene was induced in transformed larvae carrying the mouse NOS2 cDNAgene (Lowenstein et al., Proc. Natl. Acad. Sci. USA, 89:6711-6715(1992)) under the control of the heat shock promoter. NOS2 is acalcium-independent form of NOS that is capable of efficientconstitutive NO production. Imaginal discs were labelled with5-bromo-deoxyuridine (BrdU), and the extent and distribution of labelingof S-phase nuclei in leg imaginal discs from larvae after inhibition ofNOS, from NOS2 transformants after heat shock induction, and fromcontrol untreated larvae were compared. The data show that there weresignificantly more BrdU-labeled cells in imaginal discs of larvae inwhich NOS activity was suppressed by L-nitroarginine methyl ester(L-NAME) than in control untreated larvae (or larvae treated with theinactive isomer D-NAME). The data also show that there weresignificantly more BrdU-labeled cells in imaginal discs of flies inwhich NOS was inhibited than in control flies. In contrast, there weremarkedly fewer BrdU-labeled cells in imaginal discs from inducedNOS-transformed flies than in uninduced controls. At the same time,these changes in the number of BrdU-labeled cells after inhibition orectopic expression of NOS appeared to be evenly distributed over theentire disc.

These data indicate that modulation of NOS activity affects the numberof cells in S phase in imaginal discs, which is consistent with theobservations that NO suppresses DNA synthesis and cell division.

Inhibition of NOS Results in Hypertrophy of Leg Segments.

The highest levels of diaphorase staining occur during the period ofdevelopment when DNA synthesis and the rate of cell division in most ofthe imaginal disc cells slow down. The strong antiproliferativeproperties of NO and the specific pattern of diaphorase staining seen inmature imaginal discs implied that NO might act as a growth arrest agentin these structures, capable of inhibiting DNA synthesis and supportingtemporary cytostasis during the switch to metamorphosis. If NO indeedacts as an anti-proliferative agent during the late stages of larvaldevelopment, then inhibition of NOS might result in excessive growth oforgans and tissues, whereas ectopic overexpression of the NOS gene mighthave the opposite effect.

To test this hypothesis, NOS activity was inhibited by injectingspecific NOS inhibitors in the developing larvae at the end of the thirdinstar, several hours before metamorphosis. Most of the larvae completedmetamorphosis successfully, giving rise to adult flies within the normaltime frame. The resulting adults differed from normal flies in manyrespects, the most dramatic being enlargements of the appendages andother structures of the fly body. The changes included a) hypertrophy ofthe femur, tibia and the segments of the tarsus; b) overgrowth of thetissues originating from the genital disc; in extreme cases, these cellscontributed to more than one-quarter of the fly body; c) an increase ofthe overall surface of the wings; d) overgrowth of the cells of tergitesand sternites; e) hypertrophy of the humerus; f) occasional duplicationsof some areas of the eye; g) occasional realformation of genitalstructures, legs and eyes; and h) occasional ectopic formation ofmisplaced body structures.

The changes were most profound in, and most often affected the legs of,the adults. The hypertrophy was particularly strong in the third pair oflegs, where the diameter of certain segments increased 3-4 fold. Thenumber of bristles and the number of rows of bristles also increased,confirming that hyperproliferation of the cells had occurred. The legsegments most strongly affected were those (first and second tarsalsegments, tibia, and femur) whose primordia had the highest levels ofNOS at the larval and prepupal stages. The changes affected mainly theanteroposterior and dorsoventral but not the proximodistal axes, so thatthe length of the affected segments remained the same. Identical changeswere observed when two structurally unrelated inhibitors of NOS,2-ethyl-2-thiopseudourea (ETU) and L-NAME (but not D-NAME) were used,indicating that the observed effect resulted specifically from blockingNOS activity.

In summary, these data show that inhibition of NOS at the late stages oflarval development results in excessive cell proliferation and increasedsize of the structures of the body of the adult fly.

Ectopic Expression of a Mouse NOS Transgene Results in Reduced Size ofLeg Segments.

The ability of NO to inhibit DNA synthesis and cell proliferationsuggests that overexpression of NOS in developing larvae may lead todiminished cell proliferation in the imaginal discs and to a reductionin the size of organs of the adult fly. Transformed flies that expressthe mouse NOS2 transgene under the control of the heat-shock promoterwere tested. Transgenic larvae were heat-shocked within one hour afterpupariation to induce ectopic expression of NOS before the final celldivisions take place. This resulted in a reduction in the size of thelimbs of the fly. The distal segments of the legs were affected mostfrequently and to the greatest degree. In extreme cases, the wholetarsus was shortened 1.5-2 fold, and the third, fourth and fifthsegments were fused together with poorly defined boundaries. The numberof bristles in a row on the affected segments also decreased, althoughthe number of rows did not change. The segments of the adult leg mostoften affected by the overexpression of NOS (third, fourth and fifthtarsal segments) were those that were not affected by the NOS inhibitorsand whose precursors exhibited particularly low levels of diaphorasestaining in the early prepupal stages. The most terminal structures ofthe appendage, including the tarsal claw, remained intact in thesedefective legs. This suggests that the observed reduction in size wasdue to incomplete growth of the distal area of the developing appendage,rather than to complete loss of its distal structures. In contrast tothe results on NOS inhibition, the changes affected only theproximodistal axis, while the diameter of the affected segments remainedthe same. In addition to the reduction in the size of the leg segments,changes included a decrease in the overall surface of the wings, cuts inthe wings, and reduced size of tergites and sternites.

These results support the conclusion that ectopic expression of NOS atthe late stages of larval development results in a decrease in cellproliferation and a reduction in the size of the structures of the bodyof the adult fly.

Inhibition of Apoptosis Unmasks Excessive Proliferation.

In leg imaginal discs, the changes in the number of S-phase nuclei aftermanipulation of NOS activity directly correlated with the changes in thesize of the adult limbs. However, in the eye imaginal disc, an increasein the number of cells in S-phase was consistently detected afterinhibition of NOS, but the resulting adult eye usually appeared normal.The possibility that the apparently normal eye phenotype occurred as aresult of programmed cell death, which counteracts excessive cellproliferation induced by NOS inhibition and restores the normal numberof cells in the eye during metamorphosis, was tested. To suppressprogrammed cell death, GMR-P35 flies were used (Hay et al, Dev.,120:2121-2129 (1994); donated by Drs. B. Hay and G. Rubin) in whichapoptosis in the developing eye is largely prevented by expression ofrecombinant baculovirus p35 protein. p35 is a strong inhibitor ofapoptosis, which acts by inhibiting the interleukin 1B-convertingenzyme-like proteases and is able to prevent apoptosis in multiplecontexts. GMR-P35 flies express p35 under the transcriptional control ofmultimerized glass-binding site from the Drosophila Rhl promoter. Glasspromoter directs expression of the transgene in all cells in andposterior to the morphogenetic furrow in the eye disc (Ellis et al.,Dev., 119:855-865 (1993)).

When NOS was inhibited in GMR-P35 larvae, the eyes of the adult fliesshowed numerous changes, reflecting the excessive proliferation ofvarious cell types in the developing eye. The most dramatic of thesechanges was in the number of ommatidia in the adult eye, which increasedfrom the nearly invariant complement of 750 in wild type flies (747±4)and untreated GMR-P35 flies (748±6), to nearly 820 (818±21) after NOSinhibition in GMR-P35 flies. This, together with the elevated number ofcells per ommatidium, caused an increase in the overall size of the eye.Other changes in p35-expressing flies after inhibition of NOS comparedwith the control GMR-P35 flies included a) more ommatidia with anirregular shape (perhaps, because of the uneven increase in the numberof various cell types); b) more ommatidia with an irregular arrangementof the rows; and c) more ommatidia of a smaller size.

Another manifestation of the inhibition of NO production in GMR-P35flies was an increase in the number of pigment, cone, and bristle cells.Wild type ommatidia contain, in addition to eight photoreceptor cells, aset of four cone cells and two primary pigment cells, surrounded by anarray of six secondary pigment cells, three tertiary pigment cells, andthree bristles (Wolff and Ready, Pattern formation in the Drosophilaretina, in The Development of Drosophila melanogaster, M. Bate and A.Martinez-Arias, eds. (Cold Spring Harbor Laboratory Press, cold SpringHarbor, N.Y.), 1277-1326 (1993)). The number of photoreceptor andaccessory cells is normally constant, and variations in this arrangementin the eyes of the normal flies are very rare. In GMR-P35 flies, thenumber of secondary and tertiary pigment cells was increased from 12 to25 (25±4) cells per sample area (defined as described in Hay et al.,Cell, 83:1253-1262 (1995)) as a result of suppressed programmed celldeath. Inhibition of NOS in these flies resulted in a further increasein the number of secondary and tertiary pigment cells to more than 35(36±8) per sample area. This number exceeds the maximal number ofpigment cells saved from programmed cell death in untreated GMR-P35flies and suggests that extra pigment cells arise as a result ofexcessive cell proliferation caused by inhibition of NOS combined withsuppression of cell death caused by p35.

The number of ommatidia with extra primary pigment cells in GMR-P35flies after inhibition of NOS was also increased in comparison withcontrol flies, although it only slightly exceeded the levels inuntreated GMR-P35 flies. Furthermore, the number of bristles wasincreased in some areas of the eye in GMR-P35 flies after NOSinhibition, up to 4-5 per ommatidium instead of the three seen in normalflies and untreated GMR-P35 flies, and these were often mislocated.Similarly, the number of cone cells was increased from four in normaland untreated GMR-P35 ommatidia to five and six in many ommatidia ofGMR-P35 flies after NOS inhibition. Clusters of ommatidia were alsofound which contained one, two, or three cone cells, which maycorrespond to improperly formed supernumerary ommatidia that did notattain the proper set of cells.

Thus, prevention of apoptosis by baculovirus p35 protein in thedeveloping eyes of transgenic flies revealed excessive proliferation ofvarious cell types after NOS inhibition in larvae, which was otherwisemasked by programmed cell death in the larvae and pupae.

Example 2 Nitric Oxide Regulates Hematopoiesis in Animals ErythroidDifferentiation

To study the formation of cell of the erythroid lineage in the spleen ofthe irradiated recipient mice, the animals (females of F1 CBA×C57B1hybrids weighing 22-24g) were treated with 750 cGy total bodyirradiation within 3-4 hours before transplantation. This dosage wastested to be enough for complete suppression of hematopoiesis in theirradiated recipient animals. BM cells were flushed from the femurs ofsyngeneic donors and injected intravenously (10⁵ BM cells per mice) inthe recipients. The animals received twice a day injections of 100 mg/kgof L-NAME and D-NAME and 10 mg/kg of ETU for 7-10 days. Mice wereanalyzed 9-10 days after transplantation.

The differentiation status of the colonies in the spleen was evaluatedby morphological criteria and by immunohistochemical tests for thepresence of receptors to various cytokines, which are present only atspecific stages of the erythroid cells' maturation. The analysis of thecolonies in the spleen of control animals and animals treated withinactive enantiomere D-NAME (Table 1) showed that in agreement withnumerous-data, most of the colonies in the spleen (>60%) containederythroid colonies, smaller fractions contained undifferentiated blastscells (14%) or both erythroid and blast cells (13%), and small fractionsof colonies contained megakaryocytes (7.5%) and granulocytes (4%). Incontrast, when animals were treated with NOS inhibitors after BMtransplantation, most of the colonies in the spleen containedundifferentiated blast cells (up to 85% of blast cells colonies andmixed blast erythroid cells colonies). Erythroid colonies comprised only15% of the total number of colonies, and the megakaryocyte andgranulocyte colonies were not detectable. The results were similar withtwo structurally unrelated NOS inhibitors, confirming the specificity oftheir action. Thus, prolonged treatment of recipient mice after BMtransfer with NOS inhibitor, reversed the ratio of blastcells-containing colonies to the erythroid cells-containing coloniesalmost 16 fold, effectively preventing erythroid differentiation. TABLE1 Formation of hemopoietic colonies in spleens of irradiated mice afterinjection of NOS inhibitors. blast cell and blast erythroid megakarycell cells erythroid ocyte granulocyte colonies colonies coloniescolonies colonies Experiment 54.6% 30.25% 15.12% — — Control 14.3% 12.9% 61.22% 7.48% 4.08%Myeloid Differentiation

To study the formation of the cells of the myeloid lineage, celluloseacetate membranes were implanted in the peritoneal cavity of mice. After7 days, when a layer of fibroblasts had covered the membranes, the micewere irradiated as described above. BM cells from syngeneic donors wereinjected (10⁵ BM cells per mice) in the peritoneal cavity of therecipients. Animals received injections of NOS inhibitors as describedabove. Membranes with growing colonies were isolated and analyzed 7-8days later.

The differentiation status of the colonies in the spleen was evaluatedby morphological criteria, myeloperoxidase reaction, and byimmunohistochemical tests for the presence of receptors to variouscytokines, which are present only at specific stages of the myeloidcells' maturation. The analysis of the colonies on the membranes incontrol animals and animals treated with inactive enantiomere D-NAME(Table 2) showed that in agreement with numerous data, most of thecolonies (92%) contained granulocytic colonies. A much smaller fractioncontained undifferentiated blasts cells (6%), and a very small fractionof colonies contained erythroid cells (1.3%). In contrast, when animalswere treated with NOS inhibitors after BM transplantation, most of thecolonies on the membranes (up to 85%) contained undifferentiated blastcells. Colonies with differentiated cells of the granulocyte lineagecomprised only 15.6% of the total number of colonies, and a negligiblefraction of the colonies (<0.5%) contained erythroid cells. The resultswere similar with two structurally unrelated NOS inhibitors, confirmingthe specificity of their action. Thus, prolonged treatment of recipientmice after BM transfer with NOS inhibitor, reversed the ratio of blastcells-containing colonies to the granulocytic colonies almost 80-fold,effectively preventing myeloid differentiation. TABLE 2 Formation ofhemopoietic colonies on cellulose acetate in the peritoneal cavity ofirradiated mice after injection of NOS inhibitors. granulocytic blastcell colonies colonies erythroid colonies Experiment 84.46% 15.6% —Control 6.34% 92.3% 1.34%Differentiation Status of Transplanted BM Cells

To study the stage to which the transplanted cells have progressed,colonies in the spleen and on the membranes were tested with specificantibodies for receptors of various growth factors. This analysispermits one to visualize and evaluate the stage of the multistepdifferentiation process that eventually leads to erythroid or myeloiddifferentiation. We have used antibodies specific for the receptors tointerleukin 3(IL-3-R), granulocyte-macrophage colony stimulating factor(GM-CSF-R), granulocyte colony stimulating factor (G-CSF-R) anderythropoietin (EpoR). The appearance of each of these receptors marks aspecific stage in hematopoiesis.

The results of the analysis demonstrate that the blast cells in thespleen colonies (representing erythroid differentiation) haveaccumulated mostly at the stage of differentiation where they havealready acquired the receptor for IL-3, but not for erythropoietin,GM-CSF or G-CSF, whereas the colonies with morphological signs oferythroid differentiation had accumulated EpoR.

The blast cells in the colonies on the membranes (representing myeloiddifferentiation) have accumulated mostly at the stage of differentiationwhere they have already acquired the receptor for IL-3, but not forerythropoietin, GM-CSF or G-CSF, whereas the myeloperoxidase-positivecolonies with morphological signs of myeloid differentiation hadaccumulated GM-CSF-R and G-CSF-R.

Stem Cells in the Bone Marrow

To study the maturation of hematopoietic cells in the bone marrow of theirradiated recipient mice, the animals were treated as described aboveand the BM cells from the femurs of syngeneic donors were injectedintravenously (10⁵ BM cells per mice) in the recipients. The animalsreceived injections of NOS inhibitors (L-NAME, its inactive enantimereD-NAME and ETU) as described above, and mice were analyzed 7-10 daysafter transplantation.

The BM cells were tested for the presence of various growth factorreceptors which serve as markers of the differentiation stage andindicate the presence of stem cells and multipotent precursor cells. TheBM preparations were tested for cells expressing receptors to HSF(ligand of c-kit), GM-CSF, G-CSF and IL-3. The results Table 3 show thatinhibition of NO synthesis in the recipient animals after BM transferleads to dramatic increase in the number of c-kit-positive andIL-3-R-positive cells, suggesting that the population of cells in the BMbecomes highly enriched in hematopoietic stem cells. At the same timethe number of cells expressing receptors for G-CSF, which marks thelater stages of differentiation, decreases almost three-fold, while thenumber of GM-CSF-R-positive cells is slightly decreased. This suggeststhat inhibition of NOS during hematopoiesis selectively enriches the BMin undifferentiated stem cells which have already acquired c-kit and IL3receptors, but have not proceeded to the later stages when the receptorfor G-CSF is synthesized. TABLE 3 Presence of hematopoietic markers inBM cells of irradiated mice after injection of NOS inhibitors Markersc-kit IL-3-R G-CSF-R GM-CSF-R control (no injections) 2% 7% 24% 18%treatment with L-NAME 46% 58% 12% 13% treatment with ETU 84% 83% 8% 10%treatment with D-Name 7% 12% 19% 16%Reversibility of the NOS Inhibitors' Action in BM Cells

The critical question is whether undifferentiated stem cells whichaccumulate in the bone marrow as a result of treatment with NOSinhibitors have the capacity to revert to normal state and resume normalhematopoiesis process once the action of NOS inhibitors is suspended.The failure to do so might indicate that the cells become stranded intheir undifferentiated status, similar to various pathologicalconditions. To answer this question, the treatment of mice with NOSinhibitors was halted 7-9 days after the BM transfer and checked the BMcells for the presence of hematopoiesis markers 1-7 days aftertermination of injections. Control mice continued to receive the dailyinjections, The results (Table 4) demonstrate that once the treatmentwith inhibitors of NOS is suspended, the cells were able to resume theirdifferentiation and to proceed to the later stages normally. Thisindicates that enrichment in stem cells after treatment with NOSinhibitors is reversible and can be used to “boost” the number of stemcells before inducing them to proceed further along theirdifferentiation pathways. TABLE 4 Presence of hematopoietic markers inBM cells of irradiated mice after injection of NOS inhibitors andsubsequent suspension of treatment Markers c-kit IL-3-R G-CSF-R GM-CSF-Rtreatment with L-NAME for 8 78% 77% 9% 11% days treatment with L-NAMEfor 82% 84% 9% 12% 13 days 1 day after suspension of 62% 71% 15% 14%treatment 2 days after suspension of 29% 37% 16% 14% treatment 3 daysafter suspension of 9% 28% 18% 18% treatment 5 days after suspension of8% 14% 28% 21% treatment 7 days after suspension of 5% 12% 26% 20%treatmentNOS Inhibition and Apoptosis

To test whether prolonged treatment with NOS inhibitors affects the rateof programmed cell death in BM cells, the number of apoptotic cells inthe preparation of BM cells was examined. The TUNEL approach was used,thus revealing the cells with intensely fragmented DNA, a hallmark ofapoptosis, at the sane time using the DAPI staining to visualize thenuclei of all cells in the preparation. The results indicate thatneither prolonged treatment with L-NAME, nor with ETU did not affect theproportion of apoptotic cells (8±3% in control versus 7± in L-NAMEtreated and 8% ± in ETU-treated animals). Similarly, suspension oftreatment with inhibitors did not affect programmed cell death in BMpreparations (9±4% of TUNEL-positive cells). This suggests thatmanipulation of NOS activity in the animals after BMT, although havingprofound effect on differentiation and maturation of hematopoieticcells, does not affect the extent of programmed cell death in BM cells,further supporting the feasibility of applications of NOS inhibitors fortherapy.

Example 3 Nitric Oxide Regulates Brain Development in Vertebrates

It has been recently demonstrated that nitric oxide (NO), amultifinctional second messenger, is involved in cell and tissuedifferentiation and organism development. NO synthase (NOS) controls thetransition from cell proliferation to growth arrest and, as a result,regulates the balance between cell proliferation and differentiation incultured neuronal cells, in developing Drosophila, and duringhematopoiesis in mammals (Peunova et al., 1996; Kuzin et al., 1996;Michurina et al., 1997). Here, whether NOS is involved in the braindevelopment in vertebrate animals was tested. Xenopus laevis was chosenas a model organism for these studies, focusing the investigation on theformation of the brain. The Xenopus NOS gene was cloned and thedistribution of NOS-positive neurons in the developing brain wasstudied. It was found that inhibition of NOS dramatically increases thenumber of cells in the developing brain, and increases the overall sizeof the brain. The results suggest that NOS is directly involved in thecontrol of cell proliferation and neuronal differentiation in thedeveloping vertebrate brain.

Cloning of the Xenopus NOS Gene

Using the information about the known NOS genes, the NOS cDNA fromXenopus (XnNOS) was cloned. Analysis of its primary structure suggeststhat the cloned gene represents the homologue of the Ca 2⁺-dependentneuronal NOS isoform of mammals. Analysis of the gene reveals aremarkable degree of evolutionary conservation with long stretches ofamino acid sequences identical to those of humans, mice, rats, andDrosophila. The cloned gene produces enzymatically active protein whentransfected in cultured cells. The primary structure of the gene made itpossible to obtain a specific antibody, and the immunofluorescenceanalysis indicates that the diaphorase staining of the developingXenopus correctly represents the distribution of the XnNOS enzyme. Thisnotion is supported by in situ hybridization analysis of XnNOStranscripts in the tadpole brain. The cloned gene is now being used toisolate other putative NOS genes from Xenopus.

NOS is Expressed in a Consistent Spatio-temporal Pattern in theDeveloping Xenopus Brain

The Xenopus brain undergoes histogenesis starting at stage 39-40; priorto that, the neural tube consists of rapidly dividing undifferentiatedneuroepithelial cells. In the growing brain of the Xenopus tadpole, newcells arise in the narrow zone of the germinal layer in a definedpattern, which can be revealed by labeling with BrdU. The distributionof NADPH-diaphorase staining (which is indicative of NOS expression) inXenopus brain from stage 40 through stage 50 was analyzed. Zones ofstaining first appeared at stage 43, the time of migration of youngneurons off the neural tube and their differentiation. Staining appearedoutside of the germinal layer and became more intense as development oftadpoles proceeded. The most intense staining was observed in singlelarge differentiated neurons in the tectum and spinal cord, and in themarginal zone of the tectum composed of processes of differentiatedneurons. The gradient of diaphorase staining was latero-medial andreciprocal to the pattern of proliferation, suggesting that zones ofactive proliferation in the germinal layer remained free of NOS activitythrough these stages.

Inhibition of NOS in the Developing Brain Resulted in ExcessiveProliferation of Young Neurons.

To test whether NOS is involved in growth arrest in neuronal precursorsin the developing Xenopus brain, NO production was blocked byintroducing pieces of plastic impregnated with NOS inhibitors, L-NAMEand ETU, into the ventricle of the tadpole's brain at stage 43. After 3,7 and 12 days, animals were examined for changes in the patterns of celldivision, differentiation, survival, and morphology of the brain. BrdUlabeling demonstrated a dramatic increase in the number of cells in theS phase of the cell cycle in the inhibitor treated brains, compared tothe control brains. The number of BrdU-positive cells in the tectumconsistently increased throughout the experiment. Staining of cellnuclei with DAPI revealed higher number of cells in the brain sectionsin each time interval of the experiment, indicating that excessive cellsin the S phase successfully completed the cell cycle by mitosis.

Inhibition of NOS and Programmed Cell Death

Whether the inhibition of NOS and excessive proliferation of cells inthe developing brain affects the programmed death of neurons in thetectum was tested. Using the TUNEL technique to visualize the apoptoticcells in the brain, it was found that at day 3 the number ofTUNEL-positive cells was the same in both the control and inhibitortreated tectum. However, after 7 and 12 days, there were more apoptoticcells in the brains of animals which received NOS inhibitors, than incontrol animals. The increase in the number of TUNEL-positive cells isnot due to toxicity of the inhibitors, since cells continued toincorporate BrdU very effectively. Identical changes were observed withtwo structurally unrelated inhibitors of NOS, indicating that theeffects resulted specifically from blocking NOS activity. This datasuggests that excessive proliferation of cells in the tectum leads toactivation of programmed death acting to remove the surplus neurons.Alternatively, this may indicate that differentiated neurons becamedependent on NO for survival, similar to the situation in fullydifferentiated PC12 cells (Peunova et al., 1996).

Neuronal Differentiation in the Brain is Affected by Inhibition of NOS

To test whether excessive cell proliferation induced by NOS inhibitorsaffects the distribution and differentiation of neurons in the Xenopusbrain, antibodies to specific neuronal markers which have a specific andhighly reproducible pattern of expression during Xenopus developmentwere used. It was found that the distribution of neurons positive forIslet-1, N-tubulin, and N CAM was changed after inhibition of NOS. Inparticular, the neurons were displaced into the marginal zone, neuronsin the intermediate layer were more heterogeneous and with shorterbranches than in control brains, and the distinct layered structure ofthe tectum was altered. In addition, the number of Islet-l positivemotor neurons was increased after inhibition of NOS.

Inhibition of NOS Leads to Ectopic Proliferation of Neuronal Precursors.

The Xenopus brain has a fine cytoarchitecture. Groups of neighboringcells share the place and time of birth and become involved in commonlocal circuits. The position of young and mature neurons in the brain isstrictly dependent on the place of their birth, migration, and finaldifferentiation, and compose a characteristic pattern. In the brains ofanimals treated with inhibitors of NOS, it was found, in addition toextra layers of young dividing neuronal precursors, numerous ectopicsites of neuronal proliferation. Large clusters of cells were observedin atypical location, occupying the marginal zone, various areas of thetectum, the telencephalon and the hindbrain.

Inhibition of NOS Increases the Overall Size of the Brain

Inhibition of NOS activity in the brains of developing tadpoles resultedin increased number of cells in the S-phase, accompanied by a modestincrease in programmed cell death at late stages. Together, thisincreased the total number of cells in the brain and consequentlyincreased the overall size of the brain. The most affected areas are theoptic tectum and the area immediately adjacent to the ventricle wherethe impregnated piece of plastic was inserted. In cases when the sourceof the NOS inhibitor was shifted in the ventricle towards thetelencephalon or hindbrain regions in the developing brain, an increasein size of the anterior the posterior parts of the brain, respectivelywas observed.

Taken together, these results demonstrate that NO controls the number ofneurons in the developing brain, and inhibition of NOS directly affectsthe size of the Xenopus brain. This confirms the role of NO as a generalregulator of cell and tissue differentiation in the organism. Thissuggests that manipulations of the NO levels may be used for therapeuticpurposes to control proliferation and subsequent differentiation ofnerve cells in replacement therapy after neurodegenerative disorderscaused by aging (e.g., Alzheimers, Parkinson's or Huntington's), stroke,or trauma.

Example 4 Nitric Oxide and Hematopoietic Stem Cell Enrichment

Materials and Methods

Animals

Female mice were used at 8-12 week of age, and were of the followingstrains: C57B1/6, B6 CBAF1/J, CBAB6F1/J, DBA (purchased from JacksonLaboratories or Taconic Farms). All mice were bred and maintained at theAnimal Care facility of CSHL on standard food diet and acidified waterad libidium.

Irradiation and Bone Marrow Transplantation

Recipient mice were exposed to 8.2-9.5 Gy total body gamma irradiationusing Marc I irradiator from Cesium-137 source (Atomic Energy of Canada,Ottawa) at a dose rate of 1.06 Gy/min 3-20 hours before bone marrowtransplantation. The dose of irradiation is enough to suppresshematopoiesis in recipient mice. NO action on hematopoiesis was studiedby BM transfer after total body irradiation. The donor mice weresacrificed by CO2 asphyxiation or cervical dislocation and the femuresand tibiae were isolated. The bone marrow cells were extracted from thefemures and tibiae were isolated. The bone marrow cells were extractedfrom the femures and tibiae by repeatedly flushing the bones withDulbecco modified Eagle medium (DMEM) (GibcoBRL). Single cellsuspensions were prepared by drawing the bone marrow through a 21-gaugeneedle followed by a 26-gauge needle and through 70 mkm nylon cellstrainer. Cells were counted using a hematocytometer. 3-5×10⁴ nuclearbone marrow cells were injected into tail vein or 1×10⁶ cells wereinjected intraperitoneally. Spleens or testis were cut into pieces, thendrawn through a 21-gauge needle and a 70 mkm nylon cell strainer toobtain a single cell suspension.

Spleen Colony Assay

The spleen colony assay of Till and McCulloch (Till, J. E. andMcCulloch, E. A., Radiat. Res., 14:213 (1961)) was applied. 3×10⁴ bonemarrow cells were injected into lethally irradiated mice (8.5-9.5 Gytotal body irradiation from a Cesium-1 37 source at a dose a 1.06Gy/min). The spleens were removed on days 8 or 12 after transplantation,fixed in Carnua's (96% ethanol:chlorophorm:acetic acid: 6:3:1) orBouin's solution (Sigma), and macroscopically visible spleen colonieswere counted. Secondary transfer of bone marrow or spleen cellsuspensions were assayed 12 days after primary transplantation. Thenumber of day-8 and day-12 spleen colonies in secondary animals wascounted.

NOS Inhibitor Administration

N-omega-Nitro L-arginine (L-NAME) (Sigma), N-omega-Nitro D-arginin(D-NAME) (Sigma) and 2-ethyl-2-thiopseudourea hydrobromide (Calbiochem)(ETU) were used. To suppress NOS activity in the recipient animals, theywere injected intravenously or intraperitoneally with 0.3 ml of amixture of two NOS inhibitors L-nitro-methylester (L-NAME) at 300 mg/kgof body weight, and 2-ethyl-2-thiopsudourea (ETU) at 30 mg/kg of bodyweight immediately after bone marrow transplantation. Such injectionswere repeated twice a day for 3-17 days. In different experimentalgroups of animals the treatment was suspended after 3, 5, 7 or 9 days.The animals in the control group received injections of 0.3 ml of salinesolution.

FACS

To prepare cells for FACS, mice were killed by cervical dislocation andbone marrow cells from both femurs and tibiae were flushed out using a 2mL syringe with 21-gauge needle followed by 26-gauge needle. Spleens andtestis were cut into pieces, then drawn through a 21-gauge needle and a70 mkm nylon cell strainer to obtain a single cell suspension. Bonemarrow, spleen, or testis cells were counted using a hematocytometer.After washing in MEM (Minimal Essential Medium, GibcoBRL) and wassolution 3% fetal bovine serum on PBS) hemapoietic cells (bone marrow orspleen cells) were resuspended in PBS (phosphate buffered saline)containing 3% fetal bovine serum. Erythrocytes were lysed with ammoniumchloride-potassium bicarbonate buffer (154 mM ammonium dichloride, 10 mMpotassium bicarbonate, 0.082 mM EDTA) 5 minutes at room temperature.After washing, cell suspensions were filtered through a 70 mkm pore sizenylon cell strainer and were counted using a hemacytometer. 3-5×10⁶nuclear hemopoietic cells in 50 ul PBS supplemented with 3% fetal bovineserum were incubated with 50 ul antibodies for 20-40 minutes at 4° C. inthe dark. Then cells were washed twice with PBS and fixed with 300-500ul of 2% formaldehyde in PBS. For two step procedure hemopoietic cellsafter washing with PBS were incubated with second antibodies 20-30minutes in the dark, then they were washed twice with PBS and fixed with300-500 ul of 2% formaldehyde in PBS. The negative controls wereunstained cells or cells stained with only second antibodies. All cellswere kept on ice throughout the whole procedure. Fixed cells were keptin the refrigerator at 4° C. till flow cytometry analysis. Control andstained samples were analyzed using an EPICS Elite cell sorter (Coulter,Hialeah, Fla.).

Antibodies

The antibodies used in immunofluorescence staining included E13-161.7(anti-SCA-1 [Ly-6A/E]), conjugated with phycoerithrin (PE) (PharMingen),2B8 (anti-c-kit), conjugated with FITC (PharmMingen), V-18 (anti-IL-3Ralfa) (Santa Cruz Biotechnology, Inc.), M-20 (anti-EpoR) (Santa CruzBiotechnology, Inc.), M-20 (anti-G-CSFR) (Santa Cruz Biotechnology,Inc.), anti-rabbit IgG-fluorescein conjugated (FITC). Anti-nNOS mAb,anti-macNOS mnAb, anti-eNOS mAb and polyclonal anti-nNOS antibodies werepurchased from Transduction Laboratories. Polyclonal anti-nNOSantibodies were also purchased from Zymed.

BrdU Labeling

To identify cells in S phase mice were injected intraperitoneally with50 ug/ml 5-Bromo-deoxyuridine (BrdU) (Beckton-Dickinson) once a day for5 days. Bone marrow cell smears were prepared and fixed with 4%formaldehyde. BrdU-Iabeled S phase nuclei were visualized afterdenaturing DNA in 2M HCl, 0.5% Triton for 2 hours, and incubation withfluorescein-conjugated antibodies to 5-BrdU (Becton Dickinson) assuggested by the manufacturer. Samples were analyzed on a Zeiss Axiphotfluorescent microscope. For nuclei visualization, smears were stainedwith DAPI, a fluorescent DNA stain (Molecular Probes), at 1 uM.

TUNEL

Analysis of apoptosis was performed on bone marrow cell smears fixed 15minutes with 4% formaldehyde in PBS by TUNEL assay (Boerhinger Manheim)as suggested by the manufacturer.

NADPH Diaphorase

NADPH-diaphorase staining was performed essentially as described(Dawson, T. M., et al., Proc. Natl. Acad Sci. USA, 88:7797 (1991) andHope, B. T., et al., Proc. Natl. Acad. Sci. USA, 88:2811 (1991)) withminor modifications. Cells were fixed in 3.7% paraformaldehyde for 1hour, washed in PBS, and incubated for 60 min at 37° C. in the stainingsolution containing 1 mM NADPH, 0.025% Nitroblue tetrazolium salt and0.3% Triton.

Peripheral blood was analyzed using standard methods. Leukocytes werecounted in the hematocytometer and in methanol-fixed and Giemza stainedsmears of peripheral blood.

Results

Inhibition of NOS Activity in Experimental Animals

In order to increase the number of stem and early progenitor cells inthe bone marrow the following protocol was used:

-   -   a) a mixture of two NOS inhibitors, L-NAME (concentration 300 mg        per kg of body weight) and ETU (concentration of 30 mg per kg of        body weight) was introduced intraperitoneally twice a day.    -   b) the treatment was stopped after 3, 4, 5, 7 or 9 days and the        presence of specific markers was analyzed in the bone marrow by        FACS analysis 1, 2, 3, 5, 7, etc. days after the cessation of        treatment.

This protocol has dramatically increased the proportion of earlyprogenitor cells in the bone marrow. At first the increase is minimal oractually reversed compared to the control animals. However, several daysafter cessation of treatment, the proportion of progenitor cells(c-kit-positive) became much higher than in control animals whichreceived the saline solution.

The content of c-kit-positive cells in the bone marrow was increasedfrom 5.1% to 23.9%. The content of IL-receptor-positive cells wasincreased from 4.3% to 25%. The content of Sca-positive cells in thebone marrow was increased from 1.7% to 5.1%. The content of Sca- andc-kit-positive cells (Sca⁺ c-kit⁺ cells) in the bone marrow wasincreased from 0.4% to 1.48%.

The highest increase for c-kit in the bone marrow was at day 1 aftercessation of reatment with NOS inhibitors which was ongoing for 9 days.The highest increase for IL3-R in the bone marrow was at days 2-3 aftercessation of treatment with NOS inhibitors which was going for 9 days.The highest increase for Sca in the bone marrow was at days 1-2 aftercessation of treatment with NOS inhibitors, also ongoing for 9 days.

Similar changes were observed in the spleens of the treated animals. Thehighest increase for c-kit in the spleen was at days 3-4 after cessationof treatment with NOS inhibitors which was going for 9 days. The highestincrease for IL3-R in the spleen was at days 3-5 after cessation oftreatment with NOS inhibitors which was going for 9 days. The highestincrease for Sca in the spleen was at days 1-3 after cessation oftreatment with NOS inhibitors which was going for 9 days. Thus, thechanges in the content of the early progenitor markers in the spleenwere following the kinetics of maturation of hematopoietic precursorcells in the bone marrow.

Together, these results demonstrate that a novel protocol for inhibitionof NOS is especially effective for enrichment of bone marrow and spleenin early hematopoietic progenitors.

NOS Inhibitors and Changes in the Peripheral Blood

The composition of the peripheral blood in the animals after treatmentwith NOS inhibitors was followed. It was found that the content ofneutrophils dramatically increased and 5 days after terminating NOSinhibitors injection it was increased 4.5 fold compared with the controlanimals, whereas 7 days after termination, neutrophil content wasincreased 7.4 fold compared with the control. This demonstrated that theprogenitor cells whose content in the bone marrow was increased bytreatment with the mixture of two NOS inhibitors, successfully proceedthrough hematopoietic differentiation and are introduced in theperipheral blood as mature granulocytes. Importantly, immatureprecursors in the peripheral blood were not observed, which suggeststhat treatment with NOS inhibitors does not induce neoplastictransformation of the bone marrow.

NOS Inhibition and DNA Synthesis

To evaluate the effect of NOS inhibition on DNA synthesis in bone marrowincorporation of BrdU into the nuclei of bone marrow cells was tested.For this, mice were treated for 7 days with a mixture of two NOSinhibitors, L-NAME and ETU as described before, except that animals didnot receive gamma irradiation and bone marrow transplantation. For thelast 5 days of treatment animals received daily injections of BrdU at 50mg/kg intraperitoneally. Bone marrow was isolated and smears wereprepared, fixed using 4% formaldehyde and processed using anti-BrdUantibody as described in Materials and Methods. The proportion ofBrdU-labeled cells was significantly higher (up to 10 fold) compared tothe control bone marrow. This indicates that treatment with NOSinhibitors has a direct effect on DNA synthesis in the hematopoieticcells in bone marrow.

NOS Inhibition and Apoptosis

To test whether prolonged treatment with NOS inhibitors affects the rateof programmed cell death in bone marrow cells, the number of apoptoticcells in the preparation of bone marrow cells was tested. The TUNELapproach was used, thus revealing the cells with intensely fragmentedDNA, a hallmark of apoptosis, at the same time using DAPI staining tovisualize the nuclei of all cells in the preparation. The resultsindicated that prolonged treatment with the mixture of two inhibitors,L-NAME and ETU, does not affect the proportion of apoptotic cells in thebone marrow of experimental animals compared to the control group.Similarly, suspension of treatment with inhibitors did not affectprogrammed cell death in bone marrow preparations. This indicates thatmanipulation of NOS activity in the animals after BMT, although havingprofound effects on differentiation and maturation of hematopoieticcells, does not affect the extent of programmed cell death in BM cells,further supporting the feasibility of application of NOS inhibitors fortherapy.

Retransplantation of Bone Marrow and Spleen Hematopoietic Cells to theSecondary Recipients

As described herein, treating animals with NOS inhibitors after bonemarrow transfer dramatically increased the number of cells which expressstem and progenitor cells' markers. This indicates that as a result oftreatment with NOS inhibitors, bone marrow becomes enriched in stem andearly progenitor cells. However, it was possible that this procedureonly affected the levels of expression of the markers, or it increasedthe proportion of immediate progenitor cells but not of multipotenthematopoietic stem cells. To directly demonstrate that inhibition of NOSactivity results in an increase in the number of stem cells, theproportion of colony forming units in the bone marrow and spleen ofexperimental animals was tested by transferring the bone marrow orspleen cells to secondary recipients.

To obtain experimental animals, mice were irradiated at a dose 8.2-9, 0Gy and injected intravenously 3-5×10⁴ or intraperitoneally 1×10⁶ withbone marrow cells from syngeneic donors. Immediately after bone marrowtransplanting experimental mice received intravenous or intraperitonealinjections of a mixture of two NOS-inhibitors: L-NAME and ETU. NOSinhibitor injections were repeated twice daily for 9 days. Mice in thecontrol group were injected with saline solution. After 9 days,injections were suspended. Experimental and control mice were sacrificed1 or 3 days after termination of NOS inhibitor injections. To evaluatemultipotent stem cell (CFUs) and pre-CFUs content 3×10⁴ bone marrowcells or 1×10⁶ spleen cells from experimental or control mice weretransferred into secondary irradiated recipient mice intravenously. Inaddition, aliquots of bone marrow or spleen cells from the experimentalanimals were tested by FACS for the presence of c-kit or Sca-1 moleculesor both of them on the cell surface. After 8 and 12 days the secondaryrecipients were sacrificed and the hematopoietic colonies in theirspleen were counted. The number of day-12 spleen colonies was 3.5 foldgreater in mice which received bone marrow cells from experimentalanimals (primary recipients, treated with mixture of two NOS-inhibitorsfor 9 days and left untreated for 1 day) (3.5±0.22) than from thecontrol primary recipients which received saline solution (1.0±0.15). Incontrast, the number of day-8 spleen colonies was 2.9 fold less insecondary recipients which received bone marrow cells from experimentalmice (1.50±0.23), than in the secondary recipients which received bonemarrow cells from control mice (4.38±0.76). This indicates that underthese experimental conditions the number of more primitive CFUs-12 inbone marrow of primary recipients is increased, whereas the number ofmore committed CFUs-8 is decreased. The increase after 12 dayscorresponded to the increase in the number of c-kit-positive cells inbone marrow from the primary recipients as determined by FACS analysis.

When similar experiments were performed using only one of the NOSinhibitors, either ETU or L-NAME injected for 8-12 days, the number ofday-8 and day-12 colonies in the spleen of the secondary recipients wasincreased 1.7-2.5 fold, which indicates that treatment with a mixture oftwo NOS inhibitors is a) more effective than the use of either inhibitoralone, and b) leads to a specific enrichment of bone marrow populationwith more primitive stem cells (CFUs-12).

When experiments were performed with spleen cells transplanted from theprimary to the secondary recipients, the number of day-12 spleencolonies was 1.5 fold greater in the secondary recipients which receivedspleen cells from the experimental animals (primary recipients, treatedwith mixture of two NOS inhibitors for 9 days and left untreated for 3days) compared to the secondary recipients which received spleen cellsfrom mice of the control group injected with saline solution.

Together, these experiments directly demonstrate that inhibition of NOSin the bone marrow of primary recipients led to an increase in theproportion of multipotent hematopoietic stem cells (CFUs). These resultsindicate that exposure to inhibitors of NOS therapeutically used toincrease the proportion of stem cells in the bone marrow.

Furthermore, these experiments indicate that different inhibitors of NOSactivity and their combinations can be used for enrichment withhematopoietic stem and progenitor cells. Moreover, they suggest that, inaddition to bone marrow, a similar approach can be applied to stem andearly progenitor cells of blood such as umbilical cord vein blood orperipheral blood and other tissues of the organism to increase theircontent.

Equivalents

While this invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

1-22. (canceled)
 23. A method of decreasing a population of cells in Sphase in a tissue of a mammal and inducing differentiation of the cells,comprising contacting the tissue with at least one enhancer of nitricoxide.
 24. A method according to claim 23 wherein the enhancer is anenhancer of nitric oxide synthase.
 25. A method according to claim 23which results in a decrease in the size of an organ with which thetissue is associated.
 26. A method of coordinating developmentaldecisions of a cell type in a mammal, comprising introducing nitricoxide into the cell type or precursor of the cell type, therebyinhibiting proliferation of the cell type or precursor of the cell typeand inducing differentiation of the cell type or precursor of the celltype.
 27. A method of inducing differentiation in a mammalian cellpopulation comprising contacting the cell population with nitric oxideor a nitric oxide enhancer. 28-42. (canceled)